• Toxicological Research
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• v.2 no.1
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• pp.1-7
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• 1986

Procarcinogen induced DNA single-strand breaks and unschduled DNA synthesis were measured in primary rat hepatocytes culture. For DNA single-strand breaks assay, rat liver DNA was prelabeled by injection 3H-thymidine during the peak of DNA synthesis following partial hepatectomy. Hepatocytes were isolated from the rat 2 weeks after surgery by a collagenase perfusion techinique and maintained as monolayers in serum free medium on collagen-coated culture dishes. DNA sigle-strand breaks were measured by the alkaline elution techinique.

• Animal cells and systems
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• v.15 no.1
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• pp.19-27
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• 2011

Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• Fisheries and Aquatic Sciences
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• v.4 no.4
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• pp.186-191
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• 2001

Vitellogenin (VTG) and VTG mRNA induction by $estradiol-17\beta\;(E_2)$ were examined in the primary cultures of hepatocyte in the rainbow trout. Hepatocytes were precultured for 2 days, then $E_2$ was added and cultured for another 5 days. Media and hepatocytes were then analyzed by electrophoresis and Northern blotting for VTG and VTG mRNA, respectively. The hepatocytes were formed a few aggregates within 5 days without further spreading to a monolayer. Cell viability and high DNA content were maintained during the incubation. The hepatocyte culture with E2 induced a weak VTG band at a molecular weight of 175kDa on Day 2 after $E_2$ addition. The relative amount of VTG was expressed in percentage of total protein concentrations. VTG was gradually increased as $1.9\%$ on Day 2, $6.3\%$ on Day 4 and $7.3\%$ on Day 5. VTG mRNA band was detected at about 6.6 kb in the culture with $E_2$ at day 1 of culture. The level of VTG mRNA expression linearly increased with time until Day 5 (r=0.97).

• Toxicological Research
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• v.22 no.3
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• pp.301-306
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• 2006

Microcystin-LR(MC-LR), a cyanobacterial toxin produced by Microcystis aeruginosa, causes severe hepatotoxicity. Here we investigated the morphologic changes of rat hepatocyte spheroid induced by exposure of MC-LR($10^{-6}M$) in vitro. In addition, to determine the effects of such toxin in the process of hepatocyte spheroid formation, primarily isolated hepatocytes were incubated with MC-LR and the process of spheroid formation was observed. In both hepatocyte spheroid and suspension culture systems, the morphologic changes caused by MC-LR were noticible at 5 min post exposure and were characterized by the loss of microvilli, cytoplasmic vacuolation, the accumulation of lipid droplets, and blob formation. Especially, the size and numbers of blob on the cell surface were increased as the incubation time prolonged and the appearance of electron dense bodies were observed in the cytoplasm of hepatocyte at 20 min post exposure. Furthermore, bile canaliculi-like structures in the hepatocyte spheroids were slightly widened and the process of spheroids formation was inhibited in the isolated hepatocytes incubated with MC-LR. These results indicate that morphologic changes in. the hepatocyte membrane and organelles seem to be typical events in showing the MC-LR induced hepatotoxic effects and the spheroid culture method might be a useful experimental tool to evaluate hepatoxicity since it reflects the in vivo status of hepatocytes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.135-140
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• 1994

Over past decades, numerous in vitro model has been developed to investigate drug metabolism. In the order of complexity we found the isolated perfused liver, hepatocytes in co-culture with epithelial cells, hepatocytes in suspension and in primary culture and subcellular hepatic microsomal fractions. Because they can be easily prepared from both animals (pharmacological and toxicological species) and humans (whole livers as well as biopsies obtained during surgery) hepatocytes in primary culture provide the most powerful model to better elucidate drug behavior at an early stage of preclinical development such as : 1. the characterization of main biotransformation reactions. 2. the identification of phase I and phase II isozymes involved in such reactions 3. the evaluation of interspecies differences allowing the selection of a second toxicological animal species more closely related to man on the basis of metabolic profiles 4. the detection of the inducing and/or inhibitory effects of a drug on metabolic enzymes, the prediction of drug interactions 5. the estimation of inter-individual variability in biotransformation reactions. The use of hepatocytes, and in particular those obstained from humans, at an early stage of drug development allows the obtention of more predictive preclinical data and a better knowledge of drug behavior in humans before the first administration of the drug in healthy volunteers.

• Animal Bioscience
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• v.35 no.11
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• pp.1787-1799
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• 2022

Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.39 no.1
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• pp.23-26
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• 2006

Effects of sera from several fish species and insulin on the development of cultured Silurus asotus hepatocytes were investigated. Hepatocytes with high viability (95%) were obtained from the livers of male catfish by two step collagenase perfusion. Isolated hepatocytes, initially showed a typical round-shape, firmly attached to the culture dish within 24 h. In the presence of catfish serum, hepatocytes attached each other, spread well on the dish and developed into monolayer after 3-4 days of incubation. Cells within the established monolayer became polygonal in shape and their nuclei and boundaries being clearly visible under the microscope. In contrast, when incubated in FBS-supplemented or serum-free medium, cells managed to form small clusters, each made of 2-10 cells. Cells in FBS-supplemented medium further developed into larger clusters. However, these clusters failed to develope into monolayer. In addition, when insulin was deprived from culture medium, formation of monolayer also failed. From these data, it can be concluded that the presence of both catfish serum and insulin is necessary for the formation of monolayer of catfish hepatocytes and the functional role of fish serum may differ from that of insulin and can not be displaced by FBS-supplementation.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.186-192
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• 2001

A primary culture of human fetal hepatocytes was obtained through a therapeutic abortion process at 26 weeks of gestation period. More than $10^8$ cells were seeded on a plastic plate. These hepatocytes were infected with hepatitis B virus (HBV). The HBV was purified from serum of one chronic HBV carrier. Transformed hepatocytes were subcultured in a 10% FBS-supplemented medium. The morphology of the transformed cell was epithelial-like. The cells from the first pass showed signs of early proliferation and had a latent period of more than 3 months after 6-7 passages. After the rest period, the transformed cell proliferated actively and they were subcultured every three days. Transformed hepatocytes were characterized by detection of the HBV transcript by RT-PCR. The secretion of virions from transformed cells was investigated by PCR with the cell medium. Two types of virions secreted into the culture medium were examined by using the transmission electron microscope. Another approach to study the secretion of virions in to culture medium was carried out with HBV antibody. HBsAg was detected in the culture medium of transformed cells using ELISA and Western blot analyses. These data suggested that the human fetal hepatocyte cell line has been established by infection of HBV, in which this cell line secreted viral particles into the culture medium.

• Polymer(Korea)
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• v.29 no.3
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• pp.294-299
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• 2005

Thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) was covalently patterned by masked el electron beam irradiation. Introduction of PIPAAm on tissue culture polystyrene dish was confirmed by ATR-FTIR and ESCA measurements. Hepatocytes were cultured at $37^{circ}C$ on these surfaces. Cells adhered on PIPAAm-grafted domains were detached by reducing culture temperature to $20^{circ}C$. Endothelial cells were then seeded and cultured on the same surfaces. Seeded endothelial cells were selectively attached on hepatocytes detached and PIPAAm-grafted domains and could be co-cultured with hepatocytes on the same culture dishes with clear pattern. This co-culture method enabled long-term co-culture of hepatocytes with endothelial cells.